Binding of toluidine blue-myristic acid derivative to cucurbit[7]uril and human serum albumin: computational and biophysical insights towards a biosupramolecular assembly.

A new toluidine blue-myristic acid photosensitizer derivate (TBOMyr) was investigated as a design molecule to bind simultaneously to cucurbit[7]uril (CB[7]) and human serum albumin (HSA) with the aim of constructing a biosupramolecular assembly. Molecular docking and dynamics calculations revealed the main supramolecular and bio-molecular interactions of TBOMyr with the macrocycle or the protein, respectively. The addition of the negatively charged myristic acid-like tail resulted in a unique conformation of the CB[7] complex where the phenothiazine core was included in the cavity of CB[7], leaving the fatty acid portion free to interact with the protein. A favorable ternary interaction between TBOMyr, CB[7] and HSA was suggested by the calculations, and an experimental binding affinity in the order of 105 M-1 was determined for the TBOMyr@CB[7] complex with HSA. The new TBOMyr derivative could find applications in photodynamic therapy benefiting from the biosupramolecular interactions as a transport system.

Cucurbit[7]uril as a Supramolecular Catalyst in Base-Catalyzed Reactions. Experimental and Theoretical Studies on Carbonate and Thiocarbonate Hydrolysis Reactions.

Cucurbit[7]uril (CB7) catalyzes the hydrolysis reaction of bis(4-nitrophenyl)carbonate (1) but inhibits that of bis(4-nitrophenyl)thiocarbonate (2). Two relevant CB7 effects are proposed, a base-catalyst mediated by the CB7 portal and an inhibitory role attributed to the lower interaction of the thiocarbonyl group with the solvent in the host cavity, respectively.

5-HT2 Receptor Subfamily and the Halogen Bond Promise.

The binding of C-4-halogenated 1-(4-X-2,5-dimethoxyphenyl)-2-aminopropane (DOX) serotonin agonist psychedelics at all three 5-HT2 receptor subtypes is up to two orders of magnitude stronger for X = Cl, Br, or I (but not F) than when C-4 bears a hydrogen atom and more than expected from their hydrophobicities. Our docking and molecular dynamics simulations agree with the fact that increasing the polarizability of halogens results in halogen-oxygen distances to specific backbone C═O groups, and C-X···O angles, in ranges expected for halogen bonds (XBs), which could contribute to the high affinities observed. Good linear correlations are found for each receptor type, indicating that the binding pocket-ligand affinity is enhanced as the XB interaction becomes stronger (i.e., I ≈ Br > Cl > F). It is also striking to note how the linear equations unveil that the receptor's response on the strength of the XB interaction is quite similar among 5-HT2A and 5-HT2C, whereas the 5-HT2B's sensitivity is less. The calculated dipole polarizabilities in the binding pocket of the receptors reflect the experimental affinity values, indicating that less-polarizable and harder binding sites are more prone to XB formation.

A New Smoothened Antagonist Bearing the Purine Scaffold Shows Antitumour Activity In Vitro and In Vivo.

The Smoothened (SMO) receptor is the most druggable target in the Hedgehog (HH) pathway for anticancer compounds. However, SMO antagonists such as vismodegib rapidly develop drug resistance. In this study, new SMO antagonists having the versatile purine ring as a scaffold were designed, synthesised, and biologically tested to provide an insight to their mechanism of action. Compound 4s was the most active and the best inhibitor of cell growth and selectively cytotoxic to cancer cells. 4s induced cell cycle arrest, apoptosis, a reduction in colony formation and downregulation of PTCH and GLI1 expression. BODIPY-cyclopamine displacement assays confirmed 4s is a SMO antagonist. In vivo, 4s strongly inhibited tumour relapse and metastasis of melanoma cells in mice. In vitro, 4s was more efficient than vismodegib to induce apoptosis in human cancer cells and that might be attributed to its dual ability to function as a SMO antagonist and apoptosis inducer.

Coumarin-Chalcone Hybrids as Inhibitors of MAO-B: Biological Activity and In Silico Studies.

Fourteen coumarin-derived compounds modified at the C3 carbon of coumarin with an α,ß-unsaturated ketone were synthesized. These compounds may be designated as chalcocoumarins (3-cinnamoyl-2H-chromen-2-ones). Both chalcones and coumarins are recognized scaffolds in medicinal chemistry, showing diverse biological and pharmacological properties among which neuroprotective activities and multiple enzyme inhibition, including mitochondrial enzyme systems, stand out. The evaluation of monoamine oxidase B (MAO-B) inhibitors has aroused considerable interest as therapeutic agents for neurodegenerative diseases such as Parkinson's. Of the fourteen chalcocumarins evaluated here against MAO-B, ChC4 showed the strongest activity in vitro, with IC50 = 0.76 ± 0.08 µM. Computational docking, molecular dynamics and MM/GBSA studies, confirm that ChC4 binds very stably to the active rMAO-B site, explaining the experimental inhibition data.

New lead elements for histamine H3 receptor ligands in the pyrrolo[2,3-d]pyrimidine class.

This work describes the microwave assisted synthesis of twelve novel histamine H3 receptor ligands. They display pyrrolo[2,3-d]pyrimidine derivatives with rigidized aliphatic amines as warheads. The compounds were screened for H3R and H4R binding affinities in radioligand displacement assays and the most potent compounds were evaluated for H3R binding properties in vitro and in docking studies. The combination of a rigidized H3R warhead and the pyrrolo[2,3-d]pyrimidine scaffold resulted in selective activity at the H3 receptor with a pKi value of 6.90 for the most potent compound. A bipiperidine warhead displayed higher affinity than a piperazine or morpholine motif, while a naphthyl moiety in the arbitrary region increased affinity compared to a phenyl derivative. The compounds can be starting points for novel, simply synthesized histamine H3 receptor ligands.

Inclusion of Ethyl Acetoacetate Bearing 7-Hydroxycoumarin Dye by ß-Cyclodextrin and its Cooperative Assembly with Mercury(II) Ions: Spectroscopic and Molecular Modeling Studies.

The inclusion of the fluorescent organic dye, ethyl 3-(7-hydroxy-2-oxo-2H-chromen-3-yl)-3-oxopropanoate (1) by the host ß-cyclodextrin (ß-CD), and its response toward mercuric ions (Hg2+ ), was studied by UV/Vis, fluorescence, and 1 H NMR spectroscopic analyses, mass spectrometry and molecular modeling studies. 1 H NMR measurements together with molecular modeling studies for dye 1 demonstrate that it exhibits two tautomeric forms (keto and enol); however, when the dye is included into the ß-CD cavity, the enol form predominates. Moreover, by using spectroscopic and spectrometry techniques, a 1:1 stoichiometry was determined for the complexes formed between dye 1 (enol form) and ß-CD, with a binding constant (Kb1 =1.8×104 m-1 ) and for the dye 1 (keto form)-Hg2+ (Kb2 =2.3×103 m-1 ). Interestingly, in the presence of 1-ß-CD complex and mercuric ions, a ternary supramolecular system (Hg-1-ß-CD complex) was established, with a 1:1:1 stoichiometry and a Kb3 value of 4.3×103 m-1 , with the keto form of the dye being the only one present in this assembly. The three-component system provides a starting point for the development of novel and directed supramolecular assemblies.

Tracking the molecular evolution of calcium permeability in a nicotinic acetylcholine receptor.

Nicotinic acetylcholine receptors are a family of ligand-gated nonselective cationic channels that participate in fundamental physiological processes at both the central and the peripheral nervous system. The extent of calcium entry through ligand-gated ion channels defines their distinct functions. The α9α10 nicotinic cholinergic receptor, expressed in cochlear hair cells, is a peculiar member of the family as it shows differences in the extent of calcium permeability across species. In particular, mammalian α9α10 receptors are among the ligand-gated ion channels which exhibit the highest calcium selectivity. This acquired differential property provides the unique opportunity of studying how protein function was shaped along evolutionary history, by tracking its evolutionary record and experimentally defining the amino acid changes involved. We have applied a molecular evolution approach of ancestral sequence reconstruction, together with molecular dynamics simulations and an evolutionary-based mutagenesis strategy, in order to trace the molecular events that yielded a high calcium permeable nicotinic α9α10 mammalian receptor. Only three specific amino acid substitutions in the α9 subunit were directly involved. These are located at the extracellular vestibule and at the exit of the channel pore and not at the transmembrane region 2 of the protein as previously thought. Moreover, we show that these three critical substitutions only increase calcium permeability in the context of the mammalian but not the avian receptor, stressing the relevance of overall protein structure on defining functional properties. These results highlight the importance of tracking evolutionarily acquired changes in protein sequence underlying fundamental functional properties of ligand-gated ion channels.

Revealing Monoamine Oxidase B Catalytic Mechanisms by Means of the Quantum Chemical Cluster Approach.

Two of the possible catalytic mechanisms for neurotransmitter oxidative deamination by monoamine oxidase B (MAO B), namely, polar nucleophilic and hydride transfer, were addressed in order to comprehend the nature of their rate-determining step. The Quantum Chemical Cluster Approach was used to obtain transition states of MAO B complexed with phenylethylamine (PEA), benzylamine (BA), and p-nitrobenzylamine (NBA). The choice of these amines relies on their importance to address MAO B catalytic mechanisms so as to help us to answer questions such as why BA is a better substrate than NBA or how para-substitution affects substrate's reactivity. Transition states were later validated by comparison with the experimental free energy barriers. From a theoretical point of view, and according to the our reported transition states, their calculated barriers and structural and orbital differences obtained by us among these compounds, we propose that good substrates such as BA and PEA might follow the hydride transfer pathway while poor substrates such as NBA prefer the polar nucleophilic mechanism, which might suggest that MAO B can act by both mechanisms. The low free energy barriers for BA and PEA reflect the preference that MAO B has for hydride transfer over the polar nucleophilic mechanism when catalyzing the oxidative deamination of neurotransmitters.

2-Phenylaminonaphthoquinones and related compounds: synthesis, trypanocidal and cytotoxic activities.

A series of new 2-aminonaphthoquinones and related compounds were synthesized and evaluated in vitro as trypanocidal and cytotoxic agents. Some tested compounds inhibited epimastigote growth and trypomastigote viability. Several compounds showed similar or higher activity and selectivity as compared with current trypanocidal drug, nifurtimox. Compound 4l exhibit higher selectivity than nifurtimox against Trypanosoma cruzi in comparison with Vero cells. Some of the synthesized quinones were tested against cancer cells and normal fibroblasts, showing that certain chemical modifications on the naphthoquinone moiety induce and excellent increase the selectivity index of the cytotoxicity (4g and 10). The results presented here show that the anti-T. cruzi activity of 2-aminonaphthoquinones derivatives can be improved by the replacement of the benzene ring by a pyridine moiety. Interestingly, the presence of a chlorine atom at C-3 and a highly lipophilic alkyl group or aromatic ring are newly observed elements that should lead to the discovery of more selective cytotoxic and trypanocidal compounds.

The role of solvent exclusion in the interaction between D124 and the metal site in SOD1: implications for ALS.

Structural changes in the metal site of the copper-zinc superoxide dismutase (SOD1) are involved in the various mechanisms proposed for the pathogenesis of the SOD1-linked familial form of amyotrophic lateral sclerosis (ALS). Elucidating how the metal site of SOD1 can be disrupted by ALS-linked mutations is important for a better understanding of the pathogenesis of the disease and for developing more efficient treatments. Residue D124, a second-sphere ligand of the copper and zinc ions, is known from experimental studies to be essential for the integrity of the metal-site structure. In this work, we used density functional theory calculations and molecular dynamics simulations to elucidate which factors keep D124 attached to the metal site and how structural changes may disrupt the binding between D124 and the metal first-sphere ligands. The calculations show that D124 is kept attached to the metal site in a kinetic trap. The exclusion of solvent molecules by the electrostatic loop of the protein is found to create the binding of D124 to the metal site. The calculations also indicate that changes in the structure of the electrostatic loop of the protein can weaken the D124-metal site interaction, lowering the affinity of the zinc site for the metal. Destabilization of the electrostatic loop of SOD1 has been previously shown to be a common property of ALS-linked variants of the protein, but its role in the pathogenesis of SOD1-linked ALS has not been elucidated.

Synthesis, docking and pharmacological evaluation of novel homo- and hetero-bis 3-piperazinylpropylindole derivatives at SERT and 5-HT1A receptor.

A series of 3-(3-(4-(3-(1H-indol-3-yl)propyl)piperazin-1-yl)propyl)-1H-indole derivatives (3a-d and 5a-f) as homo- and hetero-bis-ligands, were synthesized and evaluated for in vitro affinity at the serotonin transporter (SERT) and the 5-HT1A receptor. Compounds 5b and 5f showed nanomolar affinities for both targets. The experimental data were rationalized according to results obtained from docking experiments. These findings are in agreement with our proposal that bis-indole derivatives can bind both targets, and might serve as leads in the quest of ligands endowed with a dual mechanism of action.

Collective and Coordinated Conformational Changes Determine Agonism or Antagonism at the Human Trace Amine-Associated Receptor 1.

The human trace amine-associated receptor (hTAAR1), a G protein-coupled receptor, has been postulated as a new target in the treatment of neuropsychiatric conditions. The mechanism associated with activation or inactivation by agonists or antagonists in hTAAR1 and other GPCRs has not yet been fully elucidated. In this study, we combined computational methods including homology modeling, docking, and molecular dynamic simulations to reveal novel conformational changes associated with agonist and antagonist interactions in hTAAR1. Our findings suggest a differential cascade of coordinated movements based on the presence of either an agonist or antagonist and primarily involving the second extracellular loop, transmembrane domain 5, and the third intracellular domains of hTAAR1. Our study provides an opportunity to predict the effects on new ligands with agonistic or antagonistic activity at hTAAR1 based on the reported conformational changes.

Extraordinary Control of Photosensitized Singlet Oxygen Generation by Acyclic Cucurbituril-like Containers.

Supramolecular control of singlet oxygen generation is incredibly valuable for several fields with broad applications and thus still challenging. However, macrocyclic inclusion complexes inherently restrict the interaction of photosensitizers with surrounding oxygen in the media. To circumvent this issue, we turned our attention in this work to acyclic cucurbituril-like containers and uncover their properties as supramolecular hosts for photosensitizers with extraordinary control of their photophysics, including singlet oxygen generation. Thermodynamic and photophysical studies were carried out showing that these acyclic containers compare very favorably to benchmark macrocycles such as cucurbiturils and cyclodextrins in terms of their binding affinities and supramolecular control of singlet oxygen generation. Acyclic container with terminal naphthalene walls offers a similar cavity to cucurbit[7]uril and the same carbonyl-lined portals for a tight binding of phenothiazinium dye methylene blue and stabilizing its singlet and triplet excited states. Thus, generation of singlet oxygen for this container is higher than for other macrocycles and even higher than the free photosensitizer. While the acyclic container with smaller terminal benzene walls, stacks over the dye through sulfur-π and π-π interactions deactivating the singlet and triplet excited states, thus showing the lowest generation of singlet oxygen out of all of the studied systems. Due to the great water solubility and biocompatibility of these systems, they possess great potential for novel applications in photocatalysis, synthesis, and biomedical fields, among others.

Peroxyl radicals modify 6-phosphogluconolactonase from Escherichia coli via oxidation of specific amino acids and aggregation which inhibits enzyme activity.

6-phosphogluconolactonase (6PGL) catalyzes the second reaction of the pentose phosphate pathway (PPP) converting 6-phosphogluconolactone to 6-phosphogluconate. The PPP is critical to the generation of NADPH and metabolic intermediates, but some of its components are susceptible to oxidative inactivation. Previous studies have characterized damage to the first (glucose-6-phosphate dehydrogenase) and third (6-phosphogluconate dehydrogenase) enzymes of the pathway, but no data are available for 6PGL. This knowledge gap is addressed here. Oxidation of Escherichia coli 6PGL by peroxyl radicals (ROO•, from AAPH (2,2'-azobis(2-methylpropionamidine) dihydrochloride) was examined using SDS-PAGE, amino acid consumption, liquid chromatography with mass detection (LC-MS), protein carbonyl formation and computational methods. NADPH generation was assessed using mixtures all three enzymes of the oxidative phase of the PPP. Incubation of 6PGL with 10 or 100 mM AAPH resulted in protein aggregation mostly due to reducible (disulfide) bonds. High fluxes of ROO• induced consumption of Cys, Met and Trp, with the Cys oxidation rationalizing the aggregate formation. Low levels of carbonyls were detected, while LC-MS analyses provided evidence for oxidation of selected Trp and Met residues (Met1, Trp18, Met41, Trp203, Met220 and Met221). ROO• elicited little loss of enzymatic activity of monomeric 6PGL, but the aggregates showed diminished NADPH generation. This is consistent with in silico analyses that indicate that the modified Trp and Met are far from the 6-phosphogluconolactone binding site and the catalytic dyad (His130 and Arg179). Together these data indicate that monomeric 6PGL is a robust enzyme towards oxidative inactivation by ROO• and when compared to other PPP enzymes.

Influence of protonation on substrate and inhibitor interactions at the active site of human monoamine oxidase-A.

Although substrate conversion mediated by human monoaminooxidase (hMAO) has been associated with the deprotonated state of their amine moiety, data regarding the influence of protonation on substrate binding at the active site are scarce. Thus, in order to assess protonation influence, steered molecular dynamics (SMD) runs were carried out. These simulations revealed that the protonated form of the substrate serotonin (5-HT) exhibited stronger interactions at the protein surface compared to the neutral form. The latter displayed stronger interactions in the active site cavity. These observations support the possible role of the deprotonated form in substrate conversion. Multigrid docking studies carried out to rationalize the role of 5-HT protonation in other sites besides the active site indicated two energetically favored docking sites for the protonated form of 5-HT on the enzyme surface. These sites seem to be interconnected with the substrate/inhibitor cavity, as revealed by the tunnels observed by means of CAVER program. pK(a) calculations in the surface loci pointed to Glu³²7, Asp³²8, His488, and Asp¹³² as candidates for a possible in situ deprotonation step. Docking analysis of a group of inhibitors (structurally related to substrates) showed further interactions with the same two docking access sites. Interestingly, the protonated/deprotonated amine moiety of almost all compounds attained different docking poses in the active site, none of them oriented to the flavin moiety, thus producing a more variable and less productive orientations to act as substrates. Our results highlight the role of deprotonation in facilitating substrate conversion and also might reflect the necessity of inhibitor molecules to adopt specific orientations to achieve enzyme inhibition.

Changes in Protonation Sites of 3-Styryl Derivatives of 7-(dialkylamino)-aza-coumarin Dyes Induced by Cucurbit[7]uril.

The incorporation of a guest, with different basic sites, into an organized system (host), such as macrocycles, could stabilize, detect, or promote the formation of a certain protomer. In this context, this work aimed to study the influence of cucurbit[7]uril (CB7) on dyes such as 7-(dimethylamino)-aza-coumarins, which have more than one basic site along their molecular structure. For this, three 3-styryl derivatives of 7-(dialkylamino)-aza-coumarin dyes (SAC1-3) were synthesized and characterized by NMR, ESI-HRMS and IR. The spectral behaviour of the SACs in the absence and presence of CB7 was studied. The results showed large shifts in the UV-vis spectrum in acid medium: a hypsochromic shift of ≈5400 cm-1 (SAC1-2) and ≈3500 cm-1 (SAC3) in the absence of CB7 and a bathochromic shift of ≈4500 cm-1 (SAC1-3) in the presence of CB7. The new absorptions at long and short wavelengths were assigned to the corresponding protomers by computational calculations at the density functional theory (DFT) level. Additionally, the binding mode was corroborated by molecular dynamics simulations. Findings revealed that in the presence of CB7 the heterocyclic nitrogen was preferably protonated instead of the dialkylamino group. Namely, CB7 induces a change in the protonation preference at the basic sites of the SACs, as consequence of the molecular recognition by the macrocycle.

Implications of differential peroxyl radical-induced inactivation of glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase for the pentose phosphate pathway.

Escherichia coli glucose-6-phosphate dehydrogenase (G6PDH) and 6-phosphogluconate dehydrogenase (6PGDH) are key enzymes of the pentose phosphate pathway, responsible for the NADPH production in cells. We investigated modification of both enzymes mediated by peroxyl radicals (ROO·) to determine their respective susceptibilities to and mechanisms of oxidation. G6PDH and 6PGDH were incubated with AAPH (2,2'-azobis(2-methylpropionamidine)dihydrochloride), which was employed as ROO· source. The enzymatic activities of both enzymes were determined by NADPH release, with oxidative modifications examined by electrophoresis and liquid chromatography (LC) with fluorescence and mass (MS) detection. The activity of G6PDH decreased up to 62.0 ± 15.0% after 180 min incubation with 100 mM AAPH, whilst almost total inactivation of 6PGDH was determined under the same conditions. Although both proteins contain abundant Tyr (particularly 6PGDH), these residues were minimally affected by ROO·, with Trp and Met being major targets. LC-MS and in silico analysis showed that the modification sites of G6PDH are distant to the active site, consistent with a dispersed distribution of modifications, and inactivation resulting from oxidation of multiple Trp and Met residues. In contrast, the sites of oxidation detected on 6PGDH are located close to its catalytic site indicating a more localized oxidation, and a consequent high susceptibility to ROO·-mediated inactivation.

Role of amino acid oxidation and protein unfolding in peroxyl radical and peroxynitrite-induced inactivation of glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides.

The mechanisms underlying the inactivation of Leuconostoc mesenteroides glucose 6-phosphate dehydrogenase (G6PDH) induced by peroxyl radicals (ROO●) and peroxynitrite (ONOO-), were explored. G6PDH was incubated with AAPH (2,2' -azobis(2-methylpropionamidine)dihydrochloride), used as ROO● source, and ONOO-. Enzymatic activity was assessed by NADPH generation, while oxidative modifications were analyzed by gel electrophoresis and liquid chromatography (LC) with fluorescence and mass detection. Changes in protein conformation were studied by circular dichroism (CD) and binding of the fluorescent dye ANS (1-anilinonaphthalene-8-sulfonic acid). Incubation of G6PDH (54.4 µM) with 60 mM AAPH showed an initial phase without significant changes in enzymatic activity, followed by a secondary time-dependent continuous decrease in activity to ∼59% of the initial level after 90 min. ONOO- induced a significant and concentration-dependent loss of G6PDH activity with ∼46% of the initial activity lost on treatment with 1.5 mM ONOO-. CD and ANS fluorescence indicated changes in G6PDH secondary structure with exposure of hydrophobic sites on exposure to ROO●, but not ONOO-. LC-MS analysis provided evidence for ONOO--mediated oxidation of Tyr, Met and Trp residues, with damage to critical Met and Tyr residues underlying enzyme inactivation, but without effects on the native (dimeric) state of the protein. In contrast, studies using chloramine T, a specific oxidant of Met, provided evidence that oxidation of specific Met and Trp residues and concomitant protein unfolding, loss of dimer structure and protein aggregation are involved in G6PDH inactivation by ROO●. These two oxidant systems therefore have markedly different effects on G6PDH structure and activity.

Oxidation of lysozyme induced by peroxyl radicals involves amino acid modifications, loss of activity, and formation of specific crosslinks.

The present work examined the oxidation and crosslinking of the anti-bacterial enzyme lysozyme (Lyso), which is present in multiple biological fluids, and released from the cytoplasmic granules of macrophages and neutrophils at sites of infection and inflammation. It is therefore widely exposed to oxidants including peroxyl radicals (ROO•). We hypothesized that exposure to ROO• would generate specific modifications and inter- and intra-protein crosslinks via radical-radical reactions. Lyso was incubated with AAPH (2,2'-azobis(2-methylpropionamidine) dihydrochloride) as a ROO• source. Enzymatic activity was assessed, while oxidative modifications were detected and quantified using electrophoresis and liquid chromatography (UPLC) with fluorescence or mass detection (MS). Computational models of AAPH-Lyso interactions were developed. Exposure of Lyso to AAPH (10 and 100 mM for 3 h, and 20 mM for 1 h), at 37 °C, decreased enzymatic activity. 20 mM AAPH showed the highest efficiency of Lyso inactivation (1.78 mol of Lyso inactivated per ROO•). Conversion of Met to its sulfoxide, and to a lesser extent, Tyr oxidation to 3,4-dihydroxyphenylalanine and diTyr, were detected by UPLC-MS. Extensive transformation of Trp, involving short chain reactions, to kynurenine, oxindole, hydroxytryptophan, hydroperoxides or di-alcohols, and N-formyl-kynurenine was detected, with Trp62, Trp63 and Trp108 the most affected residues. Interactions of AAPH inside the negatively-charged catalytic pocket of Lyso, with Trp108, Asp52, and Glu35, suggest that Trp108 oxidation mediates, at least partly, Lyso inactivation. Crosslinks between Tyr20-Tyr23 (intra-molecular), and Trp62-Tyr23 (inter-molecular), were detected with both proximity (Tyr20-Tyr23), and chain flexibility (Trp62) appearing to favor the formation of covalent crosslinks.